1. Field of the Invention
Humanized monoclonal antibodies against human von Willebrand factor, cells which produce the antibodies, and antithrombotic agents containing the foregoing antibodies as the active ingredient.
2. Background of the Invention
When subendothelium tissue is exposed due to injury, platelets flowing through the bloodstream immediately adhere to the subendothelium. This event triggers a series of platelet activation processes including platelet aggregation and release of intracellular granules, after which a thrombus is formed and bleeding stops. Thrombus formation is necessary for the physiological hemostatic mechanism. However, the thrombus can cause a number of thrombotic diseases such as myocardial infarction, angina pectoris, cerebral infarction and cerebral thrombosis.
Many anti-thrombotic agents have been developed to treat thrombotic diseases. However, many conventional antithrombotic agents have low effectiveness in clinical applications and have low thrombus-specificity, causing hemorrhaging as a side effect.
An important protein which functions at the early stage of thrombus formation is von Willebrand factor ("vWF"), in blood plasma. Hemorrhagic legions associated with the occurrence of qualitative and quantitative changes in vWF are indications of von Willebrand disease ("vWD"). Several antibodies against vWF are known: NMC-4 disclosed by Fujimura et al, J. Nara Med. Assoc., vol. 36, 662 (1985); RFF-VIIIRAG:1 disclosed by Tuddenham et al, Blood, vol. 177, no. 1, 113 (1992); and the monoclonal antibodies produced by hybridomas AJvW-1, AJvW-2, AJvW-3, and AJvW-4 disclosed by Nagano et al, PCT/JP95/02435 (incorporated herein by reference).
The present invention provides humanized antibodies based on the antibodies produced by hybridoma AJvW-2. This murine monoclonal antibody is an effective inhibitor of the physiological activity of vWF and would be desirable to use for treating thrombotic diseases. Unfortunately, the use of murine monoclonal antibodies such as those from AJvW-2 have certain drawbacks in human treatment, particularly in repeated therapeutic regimens. And mouse monoclonal antibodies tend to have a short half-life in humans and lack other important immunoglobulin functional characteristics when used in humans. More importantly, murine monoclonal antibodies contain substantial amino acid sequences that are immunogenic when injected into human patients. Numerous studies have shown that, after injection of foreign antibodies, the immune response elicited in a patient against the injected antibody can be quite strong, eliminating the antibody's therapeutic effectiveness after the initial treatment. Moreover, if mouse or other antigenic (to humans) monoclonal antibodies are used to treat a human disease, then subsequent treatments with unrelated mouse antibodies may be ineffective or even dangerous due to cross-reactivity.
While the production of so-called "chimeric antibodies" (e.g., mouse variable regions joined to human constant regions) has proven somewhat successful, significant immunogenicity problems remain. (See, LoBuglio, A. F. et al., Proc. Natl. Acad. Sci. USA, 86, 4220-4224 (1989); M. N. Saleh et al., Human Antibod. Hybridomas e: 19 (1992)).
In general, producing human immunoglobulins reactive with von Willebrand factor with high affinity would be extremely difficult using typical human monoclonal antibody production techniques. Thus, there is a need for improved forms of humanized immunoglobulins specific for von Willebrand factor that are substantially non-immunogenic in humans, yet easily and economically produced in a manner suitable for therapeutic formulation and other uses. The present invention fulfills these and other needs.